Device for non-pulsating post-column derivatization

ABSTRACT

A device for performing a post-column derivatization in connection with liquid chromatography includes a separating column, a detector, an eluent withdrawal unit, connected to an outlet of the separating column, that withdraws eluents from the separating column, a reagent supply unit, arranged between the outlet of the separating column and an inlet of the detector, that supplies reagents to the eluents withdrawn from the separating column and a pump that simultaneously pumps eluents from the eluent withdrawal unit and pumps reagents to the reagent supply unit, wherein the pumping of reagents takes place free of pulsations.

This application is the national phase of international applicationPCT/EP97/03623 filed Jul. 9, 1997 which designated the U.S.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The invention concerns a device for the application of post-column(derivatization) reactions (PCR) in liquid chromatography using auniversally applicable device for the pulsation-free addition ofreagents.

2. Description of the Related Art

Liquid chromatography (LC) is a highly sophisticated analyticalseparation technique. In general this term is understood asHigh-performance (High-pressure) liquid chromatography (HPLC). Usinghigh pressure the solutes of a sample are separated on a column filledwith special materials of small particle size (stationary phase) using aliquid solvent as mobile phase. The small particle size results in ahigh number of theoretical plates per unit of column length and providesexcellent conditions for the separation of the solutes. The resultinghigh cross-sectional resistance, however, requires the use of powerfulpumps to provide the force to drive the mobile phase through the column.These pumps are designed to cause as little pulsation as possible of themobile phase. The detection of the solutes separated is performed usingvarious procedures such as UN/VIS-, fluorescence-, conductivity- orelectrochemical-detections as well as radioactivity monitoring systemsafter supplementation of the mobile phase with a scintillation fluid forthe determination of solutes labeled with radioactive isotopes.

The application of this methodology is limited, however, by the generalweakness of the detection systems. Although the diversity of thesesystems appears to be enormous, the detection of very low concentrationsof analytes, which are often encountered in HPLC-analysis, is possibleonly in the case of specific chromophores and fluorophores. Inparticular, some of the very important groups of substances like sugars,amino acids and steroids etc. escape detection at low concentrations.Further, the lack of a generally applicable detection system, like theFID in Gas Chromatography, can be considered as a general disadvantageof Liquid Chromatography as an analytical tool.

One of the answers to these problems is the application of chemicalderivatization methods. These methods are based primarily on chemicalreactions resulting in the conversion of the analytes into chromophoresor fluorophores providing an amelioration of the sensitivity and, inaddition, a high degree of specificity since only the compound ofinterest is derivatized. Because of these advantages chemicalderivatization has found broad acceptance as an analytical tool in otherareas of chromatography.

In principle, two options exist for the application of chemicalderivatization in HPLC-analysis, i.e. derivatization of the analytebefore application of the sample material to the column (pre-columnderivatization) or derivatization of the analyte after its elution fromthe column (post-column derivatization). In most cases, depending on thespecific analytical problem, only one of these methods can be applied.The pre-column derivatization is well known and in general use. One ofthe advantages of this procedure is the fact that additional pumps arenot required; further, it allows long reaction times and it can be used,if reagent and derivative have similar light absorption properties. Thedisadvantages resulting from pre-column chemical derivatization arechanges of the separation characteristics of the analyte and thetendency of artifact development.

In many situations, however, post-column derivatization—from now oncalled “PCR” (post-column reaction)—has to be applied. This procedure isused preferentially in the “on-line” mode. The only prerequisite for theapplication of PCR is that the chemical derivatization reactions can becarried out in a reproducible manner; i.e.—although favorable—thereactions do not even have to go to completion prior to reaching thedetector system nor do they have to be defined chemically. Theadvantages offered by the PCR procedure in selectivity of reaction andspecificity in the subsequent detection are won at the expense of thesensitivity of detection; i.e. the theoretically possible detectionlimits of the available detection systems worsen under PCR-conditions bya more or less pronounced broadening of bands, which depends of thedimension and the quality of the PCR reactor used. The main cause of theloss of sensitivity is due, however, to a lack of stability of thebackground signal, which may cause a severe deterioration of thesignal/noise ratio, the decisive criterion of the limits of analytedetection.

The various parameters, which are decisive determinants of the detectionlimit of a typical PCR analysis can summarized as follows:

1. The intensity of analytical signal

2. The noise of the detector (high frequency noise)

3. The stability of the background signal (low frequency noise)

a, of the eluate

b, of the mixture of eluate and reagent

Parameters 1, 2 and 3a are relevant in principle in all processes ofHPLC analytics, though the problems related to the instrumentation canbe regarded as optimized to a great extent. Parameter 3b, however, isunique to PCR analysis and represents until now an unresolved problemrestricting the use and the potential advantages of PCR systems in aserious manner.

A variety of methods were proposed, therefore, to improve the handlingof reagent addition. Reagents, for example, were introduced into theeluate using porous hollow fiber membranes, permitting the diffusion ofthe molecules required for the derivatization of the analyte (U.S. Pat.No. 4,448,691). This method, however, is not universally applicable; itcan be used only under some very special circumstances. Customary, incontrast, is the use of pumps infusing the reagent into the effluent ofthe column via a special mixing device. In most cases only a reducedpulsation rather than a pulsation-free addition of the reagent ispossible resulting in a substantial increase of the background noise.The extent of the pulsation-related noise depends on the intensity ofthe detector specific signal properties of the reagent/eluate mixture aswell as on the difference of their individual signal intensities. Itfollows that the detection limit deteriorates with increasing intensityof the background signal of the fluid reaching the detector resulting inan amplification of the low frequency noise caused by the pulsations ofthe pump used to infuse the reagent.

The reason for the customary use of HPLC pumps with low pulsationproperties is the fact that the infusion of the reagent into thesmall-diameter capillaries carrying the effluent from the column to thedetector requires substantial forces because of the high internalpressure of the system. This pressure cannot be overcome by normalcommercially available pulsation-free pumps; this is relevant inparticular, if—as necessary in most instances—post-column reactors,further increasing internal resistance, are used to extend reactiontimes in order to optimize the derivatization reaction. An additionaldisadvantage of the standard HPLC-pumps is that their pulsatingproperties are optimized for flow rates which are substantially higherthan those required for the infusion of the reagent; furthermore, notall parts of these pumps being exposed to the reagent fluid arechemically resistant to its various components.

Frequently it is attempted to smooth pulsations by insertion ofconventional pulse dampers. This, however, is not very effective,because these dampers are designed to reduce pulsations under conditionsof extremely high pressure, i.e. prior to entry of the mobile phase intothe analytical column.

Pulsation-free syringe pumps, on the other hand, equipped with eitherhigh frequency stepping or synchronized motors, can be used for PCRpurposes only under very few special circumstances, i.e. unusually lowsystemic pressure combined with a low demand for reagent fluid, thusallowing the use of small diameter syringes. Syringes, however,providing sufficient volume for universal PCR application and full-time(8 h) operation must have large plunger diameters and, hence, requirethe buildup of high linear forces; neither the performance of the motorsnor the mechanical properties of the transmission devices of suchsyringe pumps are able to overcome the systemic pressure under thesecircumstances or, alternatively, are damaged irreversibly after only ashort period of time.

In conclusion, there is a demand for a generally applicable device tocarry out post-column derivatizations in HPLC analytics permitting apulsation-free supplementation of the column effluent with the necessaryreagents even if pressures at the point of entry to the HPLC capillarysystem exceed 10 bar (1 MPa). This device should be able to operatedfull-time (8 h working day); it should be simple in its construction,easy to handle, reliable and economic in its operation and reasonablypriced.

SUMMARY OF THE INVENTION

The invention is based on a pressure equalizing withdrawal/infusionprinciple. A pulsation-free infusion of reagent by application ofpulsation-free infusion pumps is made possibel when the pressure on theplunger of the infusion syringe is neutralized by an equal counterpressure, such that the pump has to overcome solely the frictionalresistance of the plunger in the syringe cylinder and of the reagent inthe capillary guiding the reagent into the HPLC system. Such conditionsare generated by parallel withdrawing and infusion of liquid andreagent, respectively, using a pair of syringes in a manner generatingequal pressure in both of these syringes, i.e. positioning the sites ofthe withdrawal unit and of the infusion/mixing unit in the HPLCcapillary system in close proximity, assuring virtually identical(systemic) pressure at both outlet and inlet sites, respectively.

In practice, two syringes are used and connected to a withdrawal unitand an infusion/mixing unit, respectively, which are positioned insequence between the outlet of the separation column and the inlet ofthe detector system. Pressure equalization is achieved placing theplungers of the withdrawal syringe head-on to the plunger of theinfusion/reagent syringe such that the outlets of both syringes arepointing in opposite directions and keeping the barrels of both syringesin a fixed position. Provided the diameters/surface areas of theplungers heads are of equal size then the force required to move theplungers in either direction is the same; thus, if the motor of the pumpis not engaged, neither withdrawal of eluate nor addition of reagentwill occur since both syringes are under equal pressure.

Under conditions of active reagent infusion there will be no change ofthe pressure in the HPLC capillary system. The force to be generated bythe motor of the pump is limited to the force required to overcome thefrictional resistance of the plunger in the syringe cylinder and of thereagent in the capillary guiding the reagent to the infusion/mixingunit. Since pressure equilibration between both syringes will occurautomatically, the withdrawing unit will gather passively a volumecorresponding to that of the reagent infused. No change in the overallflow rate of the system will occur; there will be only a replacement ofa part of eluate by the reagent. The control of the volume of exchangebetween eluate and reagent per unit of time is regulated preferably byan electronic programming device.

The invention has further advantages:

1. Syringes of any dimension can be used, provided they are resistant tothe pressures encountered. A syringe of 50 ml capacity, for example, issufficient to carry out analyses continuously over an 8 hour periodassuming an infusion rate of 0.1 ml/min of the reagent and a flow rateof 1 ml/min of the mobile phase in the HPLC capillary system.

2. Using suitable syringes, also aggressive reagents which may causedamage by corrosion in conventional HPLC pumps can be applied withoutany problem.

3. Furthermore, depending on the respective construction of the pump,various reagents can be introduced into the system either in paralleland/or sequentially using two or more pairs of syringes.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described with reference to FIG. 1 which is aschematic illustration of a device for performing a post-columnderivatization in connection with liquid chromatography according to theinvention.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Eluent (0) is pumped using an HPLC-pump (1) through a separation column(3) generating the pressure P1. Between HPLC-pump (1) and separationcolumn (3) samples can be applied to the system using an injection port(2).

The proposed device includes a withdrawal unit (4) connected to awithdrawal tube (5), which leads to a pressure equalizing syringe pump(6). Fixed to this pump (6) is a withdrawal syringe (2) for thecollection of the eluent withdrawn. The plunger (8 a) is positionedhead-on to the plunger (8 b) of the regent/infusion syringe (10) suchthat the eoutlets of both syringes are pointing in opposite directions.The pumping drive (9) remains in position since the pressure (P_(2b))resting on the withdrawal syringe (7) is equal to the pressure (P_(2a))resting on the reagent/infusion syringe (10). This resting pressurecorresponds to the pressure (P₂) at the location of the withdrawal unit(4) and a reagent supply unit (12), respectively; hence, the pressure P4(=P_(2a)-P_(2b)) equals zero.

Infusion of reagent is carried out by pulsation-free movement of thepumping drive (9), which has to overcome only the frictional resistanceof the plunger (8 b) and of the reagent in the supply tube, or capillary(11) guiding the reagent to the reagent supply unit (12). As aconsequence of the automatic/passive pressure equilibration between bothsyringes corresponding volumes of eluent are withdrawn simultaneously atthe withdrawing unit (4), and guided through the withdrawal tube (5)into the withdrawal syringe (7). As is required in most instances ofpost-column derivatizations a post-column reactor (13) can be applied,to extend reaction times in order to optimize the derivatizationreaction. Leaving the reactor, the effluent is guided through a detectorsystem (14) and then can be wasted (15), as done in most cases.

What is claimed is:
 1. A device for performing a post-columnderivatization in connection with liquid chromatography, comprising: aseparating column; a detector; an eluent withdrawal unit, connected toan outlet of the separating column, that withdraws eluents from theseparating column; a reagent supply unit, arranged between the outlet ofthe separating column and an inlet of the detector, that suppliesreagents to the eluents withdrawn from the separating column; a pumpthat simultaneously pumps eluents from the eluent withdrawal unit andpumps reagents to the reagent supply unit, wherein the pumping ofreagents takes place free of pulsations; at least one eluent syringe,connected to the eluent withdrawal unit and the pump by a withdrawaltube, that collects eluents pumped from the eluent withdrawal unit; andat least one reagent syringe, connected to the reagent supply unit andthe pump by a supply tube, that supplies reagents pumped to the reagentsupply unit, wherein the at least one eluent syringe and the at leastone reagent syringe operate oppositely to one another and during pumpingof reagents from the at least one reagent syringe to the reagent supplyunit, the at least on eluent syringe is passively filled with eluentsfrom the eluent withdrawal unit.
 2. The device according to claim 1,wherein the at least one reagent syringe has a volume of at least 50 mland addition of 0.1 ml/minute of reagent to 1 ml/minute of eluent allowscontinuous operation of the device for at least eight hours.
 3. Thedevice according to claim 1, further comprising a pump drive that drivesthe pump.
 4. The device according to claim 3, wherein the pump drive iselectronically controlled.
 5. The device according to claim 1, furthercomprising a plurality of reagent syringes.
 6. The device according toclaim 5, wherein the plurality of reagent syringes are arranged inparallel.
 7. The device according to claim 5, wherein the plurality ofreagent syringes are arranged sequentially.
 8. The device according toclaim 1, wherein a volume of eluents pumped from the eluent withdrawalunit equals a volume of reagents pumped to the reagent supply unit. 9.The device according to claim 1, wherein the at least one eluent syringeincludes a plunger and the at least one reagent syringe includes aplunger, and the pressure on the plunger of the at least one eluentsyringe from the eluents is equal to the pressure on the plunger of theat least one reagent syringe from the reagents.
 10. A method ofperforming a post-column derivatization in the course of liquidchromatography, comprising: utilizing the device of claim
 1. 11. Amethod of performing pulsation-free reagent supply in connection withpost-column derivatization in the course of liquid chromatography,comprising: utilizing the device of claim
 1. 12. A method of performingpost-column derivatization in connection with liquid chromatography,comprising: separating eluents in a separating column; withdrawing theeluents from the separating column to an eluent withdrawal unit;supplying reagents from a reagent supplying unit to the eluentswithdrawn from the separating column; simultaneously pumping eluentsfrom the eluent withdrawal unit and pumping reagents to the reagentsupply unit; collecting the eluents pumped from the eluent withdrawalunit in at least one eluent syringe; and passively filling the at leastone eluent syringe with eluents from the eluent withdrawal unit duringpumping of reagents from at least one reagent syringe to the reagentsupply unit.
 13. The method according to claim 12, wherein the supplyingof reagents to the eluents includes supplying 0.1 ml/minute of reagentsto 1 ml/minute of eluents for at least 8 hours.
 14. The method accordingto claim 12, wherein the pumping of reagents to the reagent supply unitincludes pumping from a plurality of reagent syringes.
 15. The methodaccording to claim 14, wherein the plurality of reagent syringes arearranged in parallel.
 16. The method according to claim 14, wherein theplurality of reagent syringes are arranged sequentially.